Obesity results from a massive expansion of white adipose tissue and recruitment of adipocyte precursor cells and is a common cause of insulin resistance and diabetes. Obesity may arise from increased fat cell size due to lipid accumulation or increased number of adipocytes arising from differentiation of preadipocyes to mature adipocytes. The processes that control adipogenesis include the coordinated expression of a complicated transcription factor network and numerous cellular and hormonal signals. We have recently identified the winged-helix forkhead transcription factor Foxa2 (Hnf-3_) to be expressed de novo in visceral and subcutaneous fat of genetic mouse models of obesity and diet-induced obesity. In these insulin resistant mice, the expression of Foxa2 correlates with serum insulin concentrations. Expression of Foxa2 in preadipocytes blocks adipocyte differentiation by directly activating the gene encoding adipocyte differentiation factor-1 (Pref-1). In addition, Foxa2 is a potent transcriptional activator of "insulin sensitizing" genes. We hypothesize that Foxa2 is a counter regulatory factor in the development of obesity and may be a "biomarker" of insulin resistance. To test this hypothesis we are proposing a series of molecular and genetic studies to elucidate the role of Foxa2 in adipocytes in vitro and in vivo. In aim 1 we will examine the role of Foxa2 in adipocyte differentiation and gene expression in vitro. In aim 2 we will generate and characterize animal models that either lack Foxa2 expression or overexpress Foxa2 in adipocytes. In aim 3 we are proposing to study two functional domains of Foxa2 that are important for its transcriptional activity. In aim 4 we will generate and characterize tissue-specific mutant "knock-in" mice that express a constitutive active form of Foxa2 in pancreatic islets, adipose tissue and liver, due to the loss of a putative Akt-dependent phosphorylation site. Lastly, we will perform a feasibility study to test if FOXA2 expression in adipose tissue of humans correlates with insulin resistance and therefore may be useful as a biomarker (aim 5). Together, this proposal will 1.) Help to establish the role of Foxa2 in adipocyte differentiation and obesity, 2.) Elucidate novel, posttranscriptional mechanisms that are important for Foxa2 activity, 3.) Investigate the link between FOXA2 adipocyte expression and obesity/insulin resistance in humans and 4) contribute to our basic understanding of transcriptional pathways that are regulated by insulin.